Toxins of the different C. botulinum serotypes are produced in culture as aggregates of neurotoxin and other non-toxic proteins non-covalently associated into a polypeptide complex (Schantz, E., Purification and characterization of C. botulinum toxins, In K. Lewis and K. Cassel, Jr. (eds.), Botulism. Proceedings of a symposium. U.S. Department of Health, Education, and Welfare, Public Health Service, Cincinnati, pp. 91-104, 1964; Sugii, S. and Sakaguchi, G., Infect. Immun. 12:1262-1270, 1975; Kozaki, S., et al., Jpn. J. Med. Sci. Biol. 28:70-72, 1974; Miyazaki, S., et al., Infect. Immun. 17:395-401, 1977; Kitamura, M., et al., J. Bacteriol. 98:1173-1178, 1969; Ohishi and Sakaguchi, Appl. Environ. Microbiol. 28:923-928, 1974; Yang, K. and Sugiyama, H., Appl. Microbiol. 29:598-603, 1975; Nukina, M., et al., Zbl. Bakt. Hyg. 268:220, 1987). Toxin complexes are described as M for medium, L for large and LL for very large. These toxin complexes vary in size from ca. 900 kD for type A LL toxin complex to ca. 300 kD for the type B M complex and type E complex, to 235 kD for type F M complex (Ohishi, I. and Sakaguchi, G., supra, 1974; Kozaki, S., et al., supra, 1974; Kitamura, M., et al., supra, 1969). According to Sugii and Sakaguchi (J. Food Safety 1:53-65, 1977), during culture the proportion of one toxin complex versus another is dependent on the growth medium and conditions. A type B culture grown in the presence of 1 mM Fe+2 produces an equal proportion of L and M complexes while the same culture grown in the presence of 10 mM Fe+2 produces predominantly M complex.
Some of the non-toxic proteins associated with the various toxin complexes have hemagglutinating abilities (Sugiyama, H., Microbiol. Rev. 44:419-448, 1980; Somers, E. and DasGupta, B., J. Protein Chem. 10:415-425, 1991). In particular, non-neurotoxic fractions of the L complexes of type A, B, C, and D have been shown to have hemagglutinating activity. Hemagglutinin fractions isolated from the different serotypes show some serological cross-reactivity. Non-toxic fractions from type A and B serotypes cross-react (Goodnough, M. and Johnson, E., Appl. Environ. Microbiol. 59:2339-2342, 1993) as do non-toxic fractions from types E and F. The non-toxic fractions of types C1 and D are antigenically identical as determined by Ouchterlony diffusion (Sakaguchi, G., et al., Jpn. J. Med. Sci. Biol. 27:161-170, 1974).
The non-toxic complexing proteins have been demonstrated to be essential for stabilization of the toxin during passage through the digestive tract (Ohishi and Sakaguchi, supra, 1974; Sakaguchi, G., et al., Purification and oral toxicities of Clostridium botulinum progenitor toxins, In Biomedical aspects of botulism, G. Lewis (ed.), Academic Press, Inc., New York, pp. 21-34, 1981). Pure neurotoxin has a peroral LD50 about 100-10,000 times lower than that of toxin complex on a weight basis (Ohishi, I., Infect. Immun. 43:487-490, 1984; Sakaguchi, G., Pharmacol. Therap. 19:165-194, 1983). Presumably, the complexing proteins protect the very labile toxin molecule from proteolytic cleavage and other types of inactivation by enzymes, acids and other components present in the gut and circulatory systems since the toxin and the complexing proteins are generally stable in low pH environments.
Analysis by SDS-PAGE has shown that type A toxin complex consists of seven different nontoxic proteins ranging in size from ca. 17 kD to 118 kD in association with a neurotoxic protein of ca. 147 kD (Goodnough, M. and Johnson, E., supra, 1993; Gimenez, J. and DasGupta, B., J. Protein Chem. 12:349-361, 1993; DasGupta, Canad. J. Microbiol. 26:992-997, 1980). Isolated type A toxin complex has a specific toxicity of 2-4xc3x97107 intraperitoneal LD50/mg in 18-22 g white mice. Specific toxicities of other C. botulinum toxin complexes are type B M complexxe2x80x944-5xc3x97107 LD 50/mg, type C1 M complexxe2x80x941-2xc3x97107 LD50/mg, type D M complexxe2x80x947-8xc3x97107 LD50/mg, type E M complexxe2x80x941xc3x97107 LD50/mg, type F M complexxe2x80x942-3xc3x97107 LD50/mg (Sugiyama, H., supra, 1980), and 8-9xc3x97106/mg for type G complex (Schiavo, G., et al., J. Biol. Chem. 269:20213-20216, 1994).
The biologically active neurotoxin of C. botulinum is a dichain molecule of ca. 150 kD in molecular weight. The molecule is composed of two fragments or chains that are termed the heavy chain (Hc, ca. 100 kD) and the light chain (Lc, ca. 50 kD) that are covalently connected by one disulfide bond (FIG. 1). The neurotoxin is synthesized by the organism as a single polypeptide called the protoxin and undergoes post-translational processing termed nicking by at least one protease to generate the two separate chains (Yokosawa, N., et al., J. Gen. Microbiol. 132:1981-1988, 1986; Krysinski, E. and Sugiyama, H., Appl. Environ. Microbiol. 41:675-678, 1981). The two chains are covalently bound through a disulfide bridge. The nicking event occurs in the culture fluid for proteolytic C. botulinum and through the activity of an added exogenous enzyme such as trypsin in non-proteolytic strains (Yokosawa, N., et al., supra, 1986; DasGupta, B., J. Physiol. (Paris) 84:220-228, 1990; Kozaki, S., et al., FEMS Microbiol. Lett. 27:149-154, 1985).
Binding to cell surface. The carboxyl terminus of botulinal heavy chain is responsible for receptor binding on the cell surface. Initial work done using tetanus toxin, which is very similar in structure to botulinum neurotoxin, showed binding to cell receptors involved a multiple step binding sequence. The ten C-terminal amino acids are essential for initial receptor recognition on the motor neuron via a low affinity binding site while a sequence in the middle of the heavy chain was responsible for higher affinity secondary binding through a different protein receptor (Halpern, J. and Loftus, A., J. Biol. Chem. 268:11188-11192, 1993).
Evidence shows that binding by type B botulinum neurotoxin occurs in a similar fashion (Nishiki, T., et al., J. Biol. Chem. 269:10498-10503, 1994). The initial binding of type B neurotoxin to synaptosomes has been shown to be related to the presence of sialic acid containing motor neuron membrane components such as gangliosides GDIa, and GT1b as well as a partially purified 58 kD protein that has been tentatively determined to be a synaptogamin isoform. There is minimal binding of the neurotoxin to the 58 kD high affinity receptor in the absence of the low affinity gangliosides. This indicates that the initial low affinity binding to gangliosides which are prevalent on the cell surface by the carboxyl-terminal amino acids is followed by a high affinity binding to the 58 kD protein by an undetermined region that is located more towards the amino terminus and possibly in the central portion of the heavy chain. Treatment of synaptosomes with proteases and or sialidase decreased binding of the neurotoxin to the synaptosomes.
Channel formation. Once the neurotoxin is bound to the motor neuron via the C-terminus end of the heavy chain, the light chain and the N-terminus of the heavy chain are endocytosed. The proteolytically active light chain is then released into the cytosol of the cell via a translocation event through the phospholipid vesicle membrane. This translocation event is driven by a sequence of amino acids contained in the N-terminal portion of the heavy chain. The predicted sequence responsible for translocation of botulinum toxin type A is from amino acids 650-681 and shows strong sequence homology to tetanus toxin amino acids 659-690 (Montal, M., et al., FEBS. Lett. 313:12-18, 1992). Both of these regions contain a high number of hydrophobic amino acid residues which presumably facilitate intercalation into lipid bilayers.
Under the acidic conditions of the vesicle, channels form in the lipid bilayer due to the N-terminal portion of the heavy chain associating into a bundle of amphipathic alpha-helices. These bundles contain four heavy chain portions that allow the light chain to enter the cytosol as evidenced by conformational energy calculations and direct visualization (Montal, M., et al., supra, 1992; Schmid, M., et al., Nature 364:827-830, 1993). There are believed to be two different conformations of the channel which may begin forming soon after binding of the C-terminal portion of the heavy chain. One conformation is a low conducting version while the second has a much greater conductance in electrochemical studies (Donovan, J. and Middlebrook, J., Biochem. 25:2872-2876, 1986). The difference in the two conformations can be explained by the fact that there is a change in pH from the physiologic condition under which the toxin initially binds and conductance is low to the lower pH values of the endocytotic vesicle where conductance is higher. The rate of conductance through channels has been shown to be highest at a pH of about 6.1 and lower at pH values closer to neutral (Donovan, J. and Middlebrook, J., supra, 1986).
Enzymatic activity in neuron/specificity for substrate. In order to describe the mechanism of botulinum neurotoxins in general, the synaptic vesicle docking cascade must be understood for it is the inhibition of the release of the neurotransmitter acetylcholine from cholinergic motor neurons which leads to the classic flaccid paralysis seen in botulinum-intoxicated muscle tissue.
The key event in the release of neurotransmitter is exocytosis of the synaptic vesicle contents through fusion of the synaptic vesicles with the phospholipid/protein-containing plasma membrane. Normally, synaptic vesicles are pre-docked on the inside of the plasma membrane through a series of docking proteins and acetylcholine molecules are exocytotically released by an increase in the intracellular Ca+2 concentration (Sxc3xcdhof, T., Nature 375:645-653, 1995). The docking proteins and their relationship to the synaptic vesicles is shown in Table 2.
The neurotoxic activity of all seven serotypes of neurotoxin is related to the fact that the light chains of botulinum toxin as well as the light chain of tetanus toxin are known to be zinc endopeptidases. The zinc binding region of the light chain of the neurotoxins is highly conserved and is very homologous among the different serotypes. It includes a region that possesses the zinc binding motif HExxH surrounded by sequences that show a lesser degree of homology. The intracellular target for each serotype is one or more of the proteins involved in docking of the acetylcholine containing vesicles to the neuronal membrane. Cleavage of the various neurotoxin substrates inhibits the docking of the vesicles with the plasma membrane and, hence, the release of the neurotransmitter into the synaptic junction. The various substrates for the seven serotypes of botulinum neurotoxin as well as tetanus toxin are shown in Table 2.
Because patients have developed immunity to treatment with type A botulinal toxin complex (Borodic, G., et al., Neurology 46:26-29, 1996), a toxin preparation that avoids that immunological problem is highly desired.
Arnon, et al. (U.S. Pat. No. 5.562,907) has described botulinum toxins combining the heavy and light chain of different botulinum toxin molecules. Weller, et al. (Neurosci. Letters 122:132-134, 1991) describes toxins comprising the light chain of tetanus toxin and the heavy chain of botulinum toxin type A.
In one embodiment, the present invention is a chimeric toxin comprising a botulinal neurotoxin heavy chain and a non-clostridial toxin chain, preferably covalently bonded. In one preferred embodiment of the present invention, the non-clostridial-toxin chain is the ricin A chain. In another preferred embodiment of the present invention, the botulinal neurotoxin heavy chain is botulinum toxin type A heavy chain.
Preferably, the covalent bond is a reducible disulfide linker, preferably the linker described in FIG. 2. Alternatively, it is a nonreducible covalent linker, preferably the linker described in FIG. 1.
In a preferred form of the invention, the toxicity is at least 3.0xc3x97103 mouse intraperitoneal LD50/mg protein. More preferably, the toxicity is at least 3.3xc3x97104 mouse intraperitoneal LD50/mg protein. Most preferably, the toxicity is at least 6.6xc3x97104 mouse intraperitoneal LD50/mg protein.
In another embodiment, the present invention is a method of creating a chimeric toxin. The method comprises isolating a botulinum toxin heavy chain and alkylating the free sulfhydryl residues of the chain and then conjugating a non-clostridial toxin chain to the alkylated botulinum heavy chain. Preferably, the alkylation of free sulfhydryl residues is via iodoacetamide.
It is an advantage of the present invention that targeted toxins are developed as reagents for treatment of muscle disorders.
It is another advantage that toxins with an increase in the duration of action is created. Therefore, the treatment is less burdensome for the patient because the patient does not have to be treated as frequently. Current therapy requires frequent exposure of the patient to the toxins and higher incidence of side effects, such as ptosis, and increase in antigen load, which could lead to immunity. The current invention provides durable therapy with fewer side effects. The therapy of the current invention is preferably long-lasting and permanent.
It is another advantage that a toxin with toxicity levels of greater than 3.3xc3x97104 and preferably 6.6xc3x97103 mouse intraperitoneal LD50/mg is created, which enables injection of low concentrations thus avoiding side effects and systemic reactions.
Other advantages, features and objects of the present invention will be apparent to one of skill in the art after review of the specification, claims and drawings.